There is a continuous need in medical practice, research and diagnostic procedures for rapid and accurate determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of hormones, drugs, narcotics, steroids, polypeptides, prostaglandins, proteins, antibodies or infectious organisms in blood, saliva, urine or other biological fluids must be determined in an accurate and rapid manner for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be determined (identified herein as a "ligand"), and receptor molecules specifically reactive with the ligand. Radioisotopes, fluorogens, chromogens, detectable beads and enzymes have been used to detect the resulting complex between ligand and receptor. One common example of specific binding reactions is an immunoassay in which an antigenic substance and specific antibody thereto react to form an immunological complex.
In recent years, the use of enzyme labels has received increased attention for specific binding assays because of various disadvantages associated with radioactive and fluorescent labels. Assays using enzyme labels include what are known in the art as competitive enzyme immunoassays (EIA) and both direct and indirect enzyme linked immunosorbent assays (ELISA). Another type of useful assay is known as an immunometric or "sandwich" assay, as exemplified in U.S. Pat. No. 4,486,530 (issued Dec. 4, 1984 to David et al). In all of these assays, either a receptor for the ligand, or a known quantity of ligand analog is labeled with a enzyme so that ligand-receptor complexes can be distinguished from unlabeled materials. Generally, the complexes are separated from uncomplexed materials using some type of immobilizing technique with or without washing or filtration.
Peroxidase is one enzyme which has been used to advantage as a label in analytical methods. Peroxidase acts on hydrogen peroxide as a substrate and can oxidize various chromogens or dye-providing materials to provide a detectable species at a rate proportional to the amount of peroxidase present. Various dye-providing materials are known in the art, including benzidine and its derivatives, and various leuco dyes.
U.S. Ser. No. 136,166 (filed Dec. 18, 1987 by McClune and Bishop, now U.S. Pat. No. 5,024,935) describes an improved dye-providing composition which is stabilized with certain polymers. The composition includes dye-providing leuco dyes dissolved in methanol which can be converted to detectable dyes in the presence of peroxidase and hydrogen peroxide. While the described assay has found significant usefulness in the art, particularly for pregnancy tests, separate wash and dye-providing solutions and steps are required for effective results. It would be desirable to eliminate steps and solutions in order to make the test more reliable and simple for the user.